The association of erythroblasts with macrophages plays a central role in the formation of distinct anatomic units called erythroblastic islands. Although these islands have been well characterized morphologically, virtually nothing is about the structural and functional basis of cellular contacts within erythroblastic islands. We have previously shown that physical contact between erythroblasts and macrophages promotes the terminal maturation of erythroblasts, leading to their enucleation in vitro. Importantly, we identified a novel protein termed "medhesin" which mediates the association of erythroblasts with macrophages. The disruption of medhesin's cell adhesion function dramatically inhibited the terminal maturation of erythroid cells. Our working hypothesis is that the medhesin-mediated attachment of erythroblasts to macrophages promotes erythroid maturation and is essential for erythroblast enucleation. We have recently determined the complete amino acid sequence of human medhesin from the macrophage cDNA library. The aim of this proposal is to characterize the molecular basis of medhesin function in erythroblast-macrophage association. Specifically, the following issues will be addressed: (1) Is medhesin-dependent erythroblast-macrophage contact sufficient for erythroblast maturation? We will investigate whether medhesin-mediated cellular contact alone is sufficient to produce erythroblastic terminal maturation or additional macrophage-derived factors are required. (2) How does medhesin-mediated contact between erythroblasts and macrophages promote terminal erythroid maturation? To define the molecular basis of medhesin function, an important requisite will be to identify the molecules with which medhesin associates in erythroblasts. This identification will be accomplished using immunoprecipitation, affinity- chromatography, and the yeast two hybrid assays. (3) Do the kinetics of medhesin synthesis, assembly, and turnover correlate with erythroblast maturation? We propose to quantify the rates of medhesin synthesis, assembly, and turnover in the soluble and particulate fractions of erythroblasts at defined stages of differentiation. The precise definition of temporally-regulated medhesin expression will provide clues to the basis of erythroblast-macrophage contact during erythropoiesis. Together, the proposed studies on medhesin may begin to reveal the molecular basis of erythroblastic island formation during mammalian erythropoiesis.